Myosin VI has a number of biochemical and biophysical properties that distinguish it from the more than 20 other classes of actin-based motors. One strikingly unique feature is its ability to move towards the pointed/minus end of actin, the direction opposite all other family members. This suggests that myosin VI has unique functions in vivo. Myosin VI has been implicated in a large number of different cellular processes, including endocytosis, motility, and intracellular localization, processes that utilize its dual abilities to move along actin filaments and to bind tightly to actin (anchor). These different functions are thought to require structural features and biophysical properties of myosin VI that differ from other myosins. However, virtually no information has been reported that demonstrates whether the intriguing and unique properties measured in vitro are important in vivo. In other words, we know much about what myosin VI can do, but little about what it actually does. The studies proposed will address this question, using the fruitfly Drosophila as a model system. So far, myosin VI is known to stabilize an actin structure involved in cellular remodeling during Drosophila spermatid development. In this process, myosin VI binds to a tissue-specific regulatory molecule, Androcam, rather than its usual regulator, Calmodulin. How this binding affects it function is unknown. Using transgenic Drosophila lines that express mutant and truncated/deleted forms of myosin VI to rescue myosin VI mutant defects has revealed that some unique properties measured in vitro may not play a key role in its ability to participate in actin stabilization in vivo. The proposed studies extend these observations, examining both spermatid development and other developmental processes in which myosin VI works to understand the structural and biochemical/biophysical features that are important in vivo in a wide variety of cellular contexts. Localization studies and measurements of dynamics of GFP tagged molecules will be performed to understand how different features mediate myosin VI participation in a wide variety of cellular functions. Measurements in vitro of biochemical and biophysical properties to determine how our manipulations, and altered regulation by Acam, affect myosin VI's properties will complement these in vivo studies. These studies will likely reveal insights into myosin VI mechanisms in vivo that will be valuable in understanding basic cellular processes and diseases in which myosin VI plays a role. For example, myosin VI is up regulated in several cancers, likely participating in motility important for metastasis. Additionally, several Human deafness syndromes and associated hypertrophic cardiomyopathy are caused by mutations in myosin VI. Studies of myosin VI promise to illuminate the underlying defects in these diseases and potentially reveal new treatment options. Studies of myosin VI are likely to result in a greater understanding of mechanisms involved in a number of basic cellular processes, such as endocytosis, sorting pathways for protein localization and cellular and intracellular motility. In addition, myosin VI has been implicated in several diseases, such as prostate and ovarian cancers, human deafness syndromes, and hypertrophic cardiomyopathy. Further understanding of how myosin VI works in vivo is likely to enhance our understanding of disease mechanisms and provide for new routes of treatment.